Bone metastasis targeting remains largely unexplored. Some of the bone diseases are seldom cured just because of poor distribution of drug to the bone. Zoledronic acid (ZOL) possess a strong affinity towards bone, and hence its utility in bone metastasis management makes it a perfect ligand for bone targeting. Recent studies revealed that ZOL in combination with docetaxel (DTX) showed significant synergism in the management of bone metastasis. DTX-loaded ZOL-conjugated polyethylene glycol (PEG)ylated polybutyl cyanoacrylate (PBCA) NPs (PBCA-PEG-ZOL) were prepared using the anionic polymerization technique. Physiochemical Characterization, pharmacokinetics, in vitro bone binding assay, quantitative cellular uptake, NP uptake route characterization, and cellular IPP/ApppI (isopentenyl pyrophosphate/triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-3-enyl) ester) levels were performed. DTX was efficiently entrapped (75.94 ± 3.82%) in the Zol conjugated NPs, estimated by HPLC. They had discrete spherical shape, and size of around 82 nm estimated by zetasizer and transmission electron microscopy. Biodistribution studies using technetium-99m radiolabeling showed prolonged blood circulation half-life, and that the ratio of PBCA-PEG-ZOL NPs in tumor bearing bone to the normal bone was 3 fold, at any time point. Further, ZOL conjugated NPs localization in tumor bearing bone significantly increased with time and found to be 7.5 (p<0.01), 20 (p<0.001) and 155 (p<0.001) times higher after 1, 4 and 24 h respectively, as compared with unconjugated but pegylated NPs, possibly due to the remodeling of the bone lining by tumor metastasis. In vitro bone binding assay using human simulated bone-hydroxyapatite powder results confirmed that ZOL has strong binding affinity to bone, and maintained the affinity even when used as a surface ligand conjugated to NPs. It was noticed that after 4 h of treatment with BO2 cells, ZOL conjugated NPs showed two times longer residence time (measured by flow cytometry) than un-conjugated ones. PBCA-PEG-ZOL NPs showed an enhanced cytotoxic effect (MTT assay) in both BO2 and MCF-7 cell lines, due to cell cycle arrest and apoptosis (by flow cytometry). Uptake route characterization studies (by confocal microscopy & flow cytometry) with different inhibitors revealed that PBCA-PEG-ZOL NPs uptake is not entirely based upon clathrin or caveolae mediated endocytosis. PBCA-PEG-ZOL NPs blocked the mevalonate pathway and showed 7 and 5.3 times increase in IPP and ApppI production (measured by Liquid chromatography-mass spectrometry; LC-MS), in comparison to ZOL treatment, and 138 times higher than the control group in MCF-7 cell line. These results provide evidence that ZOL-conjugated NPs provide an efficient and targeted delivery system for bone metastasis. Hence, these NPs present a promising treatment in the near future for bone related diseases.
Thanks to SAIA for providing scholarship to Vinoth Khandelwal for visiting Slovakia.
[1] G.R. Mundy, Mechanisms of bone metastasis, Cancer 80 (8) (1997) 1546–1556.
[2] E. Shane, Evolving data about subtrochanteric fractures and bisphosphonates, N. Engl. J. Med. 362 (19) (2010) 1825–1827.
[3] R.S. Weinstein, P.K. Robertson, S.C. Manolagas, Giant osteoclast formation and long-term oral bisphosphonate therapy, N. Engl. J. Med. 360 (1) (2009) 53–62.
[4] T. Chen, J. Berenson, R. Vescio, R. Swift, A. Gilchick, S. Goodin, P. LoRusso, P. Ma, C. Ravera, F. Deckert, H. Schran, J. Seaman, A. Skerjanec, Pharmacokinetics and pharmacodynamics of zoledronic acid in cancer patients with bone metastases, J. Clin. Pharmacol. 42 (11) (2002) 1228–1236.
[5] W.J. Thompson, D.D. Thompson, P.S. Anderson, G.A. Rodan, Polymalonic acids as boneaffinity agents, EP 0341961, 1989.
[6] L. Gil, Y. Han, E.E. Opas, G.A. Rodan, R. Ruel, J.G. Seedor, P.C. Tyler, R.N. Young, Prostaglandin E2–bisphosphonate conjugates: potential agents for treatment of osteoporosis, Bioorg. Med. Chem. 7 (5) (1999) 901–919.
[7] W.C. Shakespeare, C.A. Metcalf III, Y. Wang, R. Sundaramoorthi, T. Keenan, M. Weigele, R.S. Bohacek, D.C. Dalgarno, T.K. Sawyer, Novel bone-targeted Src tyrosine kinase inhibitor drug discovery, Curr. Opin. Drug Discov. Devel. 6 (5) (2003) 729–741.
[8] H. Hirabayashi, T. Takahashi, J. Fujisaki, T. Masunaga, S. Sato, J. Hiroi, Y. Tokunaga, S. Kimura, T. Hata, Bonespecific delivery and sustained release of diclofenac, a non- steroidal anti-inflammatory drug, via bisphosphonic prodrug based on Osteotropic Drug Delivery System (ODDS), J. Control. Release 70 (1–2) (2001) 183–191.
[9] F. Hosain, R.P. Spencer, H.M. Couthon, G.L. Sturtz, Targeted delivery of antineoplastic agent to bone: biodistribution studies of technetium-99m-labeled gembisphosphonate conjugate of methotrexate, J. Nucl. Med. 37 (1) (1996) 105–107.
[10] M.D. Fancis, I. Fogelman, 99mTc diphosphonate uptake mechanism on bone, in: I. Fogelman (Ed.), Bone Scanning in Clinical Practice, Springer-Verlag, New York, 1987, pp. 7–17.